Checkpoint blockade antibodies targeting PD-1 have demonstrated improved survival in metastatic head and neck squamous cell carcinomas (HNSCC) patients by reactivating effector T cells that have infiltrated the tumor microenvironment. However, PD-1 blockade still has low overall response rates approximating 18%, suggesting that the different treatment outcomes are due to intrinsic differences in the patients' diseases, such as tumor microenvironments. Recently, we have developed a new class of radioenhancers, nanoscale metal- organic frameworks (nMOFs), that can alter the immune microenvironment. Constructed via coordination between hafnium-oxo clusters and porphyrin-like molecules, nMOFs generate both hydroxyl radicals and singlet oxygen in a process termed radiotherapy-radiodynamic therapy (RT-RDT). The objective in this application is to define the mechanisms by which RT-RDT and nMOF-enabled immunotherapy alter the immune microenvironment in order to sensitize HNSCCs to checkpoint blockade. Our central hypothesis is that nMOFs can deliver the CpG oligodeoxynucleotides and synergize with RT-RDT-induced antigen release and Type I IFN expression, which stimulates CD8+ and CD4+ T cell proliferation and infiltration into HNSCCs to regress both irradiated and non-irradiated tumors treated with PD-1/PD-L1 blockade. The goal for this proposed research is to identify a novel therapy and define the mechanisms by which it alters the immune microenvironment to sensitize HNSCCs and possibly other cancers to current clinical immunotherapies. This project will use innovative molecularly tunable nMOFs having unprecedented radioenhancement via the unique RT-RDT mechanism. This proposal is significant because it addresses an unmet need of treating radioresistant and metastatic HNSCCs both directly via RT-RDT and by acting as an immunostimulant to enhance the efficacy of existing checkpoint inhibitors. This proposal will test the central hypothesis by pursuing four specific aims: (1) define the cellular mechanisms of innate immune activation after RT-RDT; (2) determine how RT- RDT affects the tumor microenvironment in squamous cell cancers; (3) evaluate the contributions of different immune components on the efficacy of RT-RDT and immunotherapy combinations; and (4) determine effective therapies for HNSCCs resistant to PD-1/PD-L1 blockade. Aim 1 will treat cells and ex vivo stimulated or cultured immune components with nMOFs and radiation to determine how RT-RDT initiates STING and Type I interferon signaling in the tumor microenvironment. Aim 2 will determine how the tumor microenvironment and extracellular matrix are affected by nMOF-mediated RT-RDT. Aim 3 will evaluate the contribution of different immune components to the anticancer efficacy of nMOFs. Aim 4 will use primary oral tumor models that are resistant to PD-1/PD-L1 blockade as a model to identify novel immunotherapy combinations that synergize with RT-RDT. Ultimately, this project will afford new therapeutic strategies using clinically relevant nanomedicines to enhance both the radiation therapy and immunological rejection of HNSCCs.